Macrophage Fey receptors (FcyR) are important in host defense and autoimmunity. Following their clustering by IgG complexes, the internalization of macrophage FcyR and their IgG ligand can proceed by several pathways. Receptor clustering by small IgG aggregates leads to internalization via endocytosis, and clustering via large particulate complexes (e.g. IgG coated cells) leads to internalization via phagocytosis. Although both processes involve activation of identical receptors by the same ligand (theFc portion ofIgG), we have observed profound differences in the molecular mechanisms mediating these internalization pathways leading to distinct functional outcomes. For example, the C-terminal tyrosine (Y298), of the FcyRIIA ITAM is critical for optimal phagocytosis, but is dispensable for endocytosis. By contrast, the N-terminal ITAM tyrosine (Y282) appears essential for both processes. Also, while phosphorylation of tyrosines in the ITAM by Src family kinases (SRTKs) is an essen- tial step for initiation of phagocytosis, our evidence indicates that endocytosis is not dependent on receptor tyrosine phosphorylation by Src kinases, nor does it involve Syk kinase. We have also observed that ubiquitination is not required for the initial step of FcyRIIA mediated phagocytosis, but is essential for FcyRIIA mediated endocytosis. Further, mutation of the FcyRIIA Y282XXL leucine (L) (Y282 intact) completely inhibits endocytosis by FcyRIIA, suggesting that both Y282 and L.285 are required for this process. This observation argues in favor of interaction of Y282XXL with the clathrin adaptor AP-2as the mechanism underlying receptor mediated endocytosis, since YXXL motifs have been implicated in clathrin-mediated endocytosis. Using cellular and molecular biology approaches, we will pursue our long term goal to define the molecular mechanism(s) underlying Fey receptor mediated endocytosis and phagocytosis. Specifically, we will determine: 1) if a FcyRIIA sequence constitutes a binding site for AP-2, 2) which ubiquitin ligases are important in FcyRIIA mediated endocytosis and phagocytosis, 3) the potential role of the inhibitory receptor Pc/RUB in regulating endocytosis, 4) the role of lipid rafts in FcvRIIA mediated phagocytosis, 5) sequences responsible for Fc/RIIA lipid raft localization, and finally 6) the role of ubiquitination in phagosomal maturation. Since Fey receptor mediated phagocytosisand endocytosisof IgG complexes play an important role in host defense and autoimmune disorders, it is essential to understand the distinct molecular mechanisms involved in these processes.